16S rRNA-Seq of the 15m submerged microbialite
Genomic DNA extraction from the samples was performed with the DNeasy PowerLyzer Power Soil Kit (QIAGEN, Hilden, Germany) following the manufacturers instructions. 16S rRNA coding regions of the extracted DNA were amplified by polymerase chain reaction (PCR) with universal EMP (Earth Microbiome Project) primer set including 515FB (GTGYCAGCMGCCGCGGTAA) and 806RB (GGACTACNVGGGTWTCTAAT) that encodes V4 region and targets nearly all bacterial and archaeal taxa (Caporaso et al., 2018). PCR mixtures were prepared including 0.8X PCR Master Mix (PlatinumTM Hot Start PCR Master Mix (2X), Thermo Fisher Scientific, Invitrogen, Eugene OR, USA), 0.2 M of each forward and reverse barcoded primers, 1.0 L of template DNA, Phusion High-Fidelity DNA polymerase (Thermo Fisher Scientific, Invitrogen, Eugene OR, USA), and DNA-free grade water up to a total reaction volume of 25 L. PCR was performed starting with a single cycle of 94  for 3 min, 35 cycles of 94  for 45 s, 50  for 60 s, 72  for 90 s, and final single cycle of 72  for 10 min. Sequencing libraries were constructed with adaptor sequences and reaction conditions from TruSeq DNA LT Sample Prep Kit and sequenced on an Illumina iSeq 100 sequencer (Illumina, San Diego, CA, USA). The reads were filtered and trimmed to a minimum Phred quality score of 10. Following this step, the reads were merged, de-replicated, and clustered into operational taxonomic units (OTUs; 97% similarity) in QIIME2 (Bolyen et al., 2019) using SILVA NR99 v.138 as the reference database (Quast et al., 2013).